Chimeric Recombinant Antigens of Toxoplasma Gondii

ABSTRACT

The invention described herein relates to a method for combining antigen fragments of  Toxoplasma gondii  proteins, in the form of chimeric fusion products, and their use as diagnostic and immunogenic agents.

FIELD OF THE INVENTION

The invention described herein relates to the technical field of thepreparation of diagnostic means not applied directly to the animal orhuman body. The invention also furnishes compounds, methods for theirpreparation, methods for their use and compositions containing them,which are suitable for industrial application in the pharmaceutical anddiagnostic field, particularly for the detection and diagnosis ofToxoplasma gondii infections, as well as for the treatment andprevention of said infections.

BACKGROUND OF THE INVENTION

Early diagnosis is a priority and highly desirable objective in allfields of medicament, particularly because it allows an appreciableimprovement in the patient's life and a concomitant saving on the partof health care systems or on the part of the actual patients. In theparticular case of the invention described herein, early diagnosis isthat of potential or existing Toxoplasma gondii infection in pregnantwomen, with particular concern for the health of the foetus, and ininfected subjects, particularly those with impaired immunity.

Toxoplasma gondii is an obligate intracellular parasite that infects allmammalian cells, including those of human subjects (McCabe andRemington, N. Engl. J. Med. 1988, 318-313-5). Morphologically, theparasite exhibits three distinct forms of infection: tachyzoite(asexual), bradyzoite (in tissue cysts, asexual) and sporozoite (inoocysts, sexual reproduction). Transmission typically occurs throughingestion of undercooked meat harbouring tissue cysts or vegetablescontaminated with oocysts shed by cats. Human infection is generallyasymptomatic and self-limiting in immunocompetent hosts. In contrast, insubjects with impaired immunity (particularly those affected by AIDS),toxoplasmosis is a severe opportunist infection, which may give rise toencephalitis with very serious outcomes (Luft, B. J., Remington J. S.,1992, Clin. Infect. Dis. 15, 211-22). Moreover, contracting primaryinfection during pregnancy may lead to miscarriages or to severe foetaldisease in mammals.

For an extensive overview of the problem of toxoplasmosis the reader isreferred to the specific medical literature.

Diagnosis of T. gondii infection is established by isolating themicro-organism in the blood or body fluids, identifying the parasite intissues, detecting specific nucleotide sequences with PCR, or detectingspecific anti-T. gondii immunoglobulins produced by the host in responseto the infection (Beaman et al., 1995 Principles and Practice ofInfectious Diseases 4th Ed., Churchill Livingstone Inc., New York,2455-75; Remington J S et al. 1995, Infectious Diseases of the Fetus andNewborn Infant, W.B. Saunders, Philadelphia, Pa., 140-267).

Main challenges for clinicians are the diagnosis of primary T. gondiiinfections in pregnant women and the diagnosis of congenital infectionin their newborns/infants. In both cases, to implement suitabletherapies in good time and to avoid possible damage to the foetus andnewborns/infants, it is very important to establish if the parasiticinfection has been contracted before or after conception in pregnantwomen. Moreover, it is essential determining when the verticaltransmission from the mother to the foetus occurred. Finally, for theclinical management of newborns/infants there is an urgent need of asensitive diagnostic method than can discriminate, early in their life,between infected and uninfected subjects, both born to mothers withprimary toxoplasmosis in pregnancy.

Seroconversion during gestation and diagnosis of congenital infection inneonates are generally done by attempting to detect the presence of thevarious classes of anti-Toxoplasma immunoglobulins (IgG, IgM, IgA,avidity of IgG), and to compare the immunological profiles of the motherversus her child. However, the available commercial assays do notprovide enough sensitivity and specificity to allow a correct diagnosisof infection in all patients. Therefore the availability of specific,sensitive and innovative diagnostic agents is desirable.

T. gondii antigens have long been known and available, first of all asantigen mixtures obtained in various ways (FR 2,226,468, Mérieux; SU533376, Veterinary Research Institute; JP 54044016, Nihon ToketsuKanso), then as subsequent isolations of pure antigens (EP 0 082 745,Mérieux; EP 0 301 961, INSERM, Pasteur; WO 89/5658, Transgene) and theircharacterization both as proteins, and of their respective genes (WO89/08700, U. Leland, Dartmouth Coll.; U.S. Pat. No. 4,877,726, Res.Inst. Palo Alto; WO 89/12683, INSERM, Pasteur; EP 0 391 319, MochidaPharm.; IT 1,196,817, CNR; EP 0 431 541, Behringwerke; WO 92/01067,CNRS; WO 92/02624, U. Flinders; WO 92/11366, Innogenetics, SmithklineBeecham; U.S. Pat. No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR2702491, INSERM, Pasteur; WO 96/02654, bioMeriéux, Transgene; EP 0 710724 Akzo; EP 0 724 016, bioMeriéux; EP 0 751 147, Behringwerke; U.S.Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300, Innogenetics;U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto;WO 99/32633, Heska; JP 11225783, Yano; WO 99/61906, Abbott; WO 99/66043,Smithkline Beecham; JP 2000300278, Yano; WO 00/164243, Virsol), andfinally, the isolation and characterization of the antigenic regions ofToxoplasma gene products (WO 03/080839, Kenton S. r. l.)

Numerous studies have found various different antigenic proteins of T.gondii and the gene sequences of these have also been determined.

Among the most interesting proteins both for diagnostic and therapeuticpurposes, in the form of vaccines, we should mention: the micronemeproteins (WO 03/080839, Kenton S. r. l.; Beghetto et al., The Journal ofInfectious Diseases, 2005, 191:637-645; Beghetto et al, InternationalJournal for Parasitology, 2003, 33:163-173); the surface antigens SAG1(or P30) (WO 89/08700, Stanford University; WO 89/12683 Pasteur, INSERM;WO 94/17813, WO 96/02654, Transgene, bioMeriéux; EP 0 724 016, WO99/61906, U.S. Pat. No. 5,962,654, Harning et al., Clinical andDiagnostic Laboratory Immunology, May 1996, 355-357) and SAG2 (or P22)(Parmley et al., 1992, J. Clin. Microbiol. 30, 1127-33); the densegranule proteins GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO89/05658, Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86,7537-41), GRA2 (or P28) (WO 93125689, INSERM, Pasteur; U.S. Pat. No.5,633,139, U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst.Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3-14), GRA4(Mevelec et al., Mol. Biochem. Parasitol 56, 227-38), GRA6 (or P32) (FR2,702,491, INSERM, Pasteur; Lecordier al, Mol. Biochem. Parasitol. 70,85-94), GRA7 (WO 99/61906, Abbott; Jacobs et al., Mol. Biochem.Parasitol. 91, 237-49) and GRA3 (Robben et al. 2002, J. Biol. Chem. 277,17544-47): the rhoptry antigens ROP1 (or P66) (U.S. Pat. No. 5,976,553,U. Leland; EP 0 431 541, Innogenetics) and ROP2 (or P54) (Sharma et al.,J. Immunol., 131, 377-83).

As described in the above-mentioned references, the antigens wereobtained with recombinant cDNA techniques in expression vectors. Forexample, for the antigen SAG1, WO 98/08700 uses known expression vectorsin phage lambda-gt11. WO 98/12683 uses the same phage and transfects E.coli with a proprietary plasmid, or by preparing a special expressioncassette, as in WO 96/02654. EP 0 724 016 obtains mimotopes usingcombinatorial expression libraries of peptides. EP 0 301 961 describeshow to obtain excretion-secretion antigens with molecular weightsranging from 20 kDa to 185 kDa. WO 89/05658 describes a proteincontaining the epitopes of the 24 kDa protein recognized by theantibodies produced against Toxoplasma excretion-secretion antigens;this protein is obtained by transfection of cells by means of expressionvectors. WO 03/080839 describes a method based on phage-displaytechnology for identifying antigen fragments of T. gondii proteins andtheir use as diagnostic and immunogenic agents. The antigen P28 (GRA2)is described in U.S. Pat. No. 5,633,139 and the method of obtaining itis again implemented through expression in phage lambda-gt11. Theantigen P32 (GRA6) is described in patent FR 2,702,491, the antigen ROP1(P66) in U.S. Pat. No. 5,976,553, P35 (or GRA8) in EP 0 431 541, WO99/57295 and WO 99/61906, and lastly P68 in EP 0 431 541.

Yang et al. (Parasitol. Res., 2004, 92: 58-64) describe a chimericprotein containing SAG1 and SAG2 and its use to develop immunity againstT. gondii in mice.

Chinese Patent 11 94991C discloses a recombinant fusion proteincontaining two toxoplasma antigens (GRA6 and p30). No data are reportedto show that assays based on this recombinant fusion protein display therequired sensitivity in IgG- and IgM-based tests.

During the last ten years, several studies have reported the use ofrecombinant antigens for the serological diagnosis of T. gondiiinfection. Nevertheless, although promising none of the assays based onrecombinant antigens displayed all the characteristics required toreplace the tachyzoite antigen in IgG- and IgM-based tests, indicatingthat further work is needed before an immunoassay employing recombinantproducts will be available for clinical purposes.

Thus the main aim of the studies in this filed is to improve theperformance of enzyme-linked immunoassays based on recombinant products,thus improving, for example early diagnosis of congenital toxoplasmosisin newborns/infants.

SUMMARY OF THE INVENTION

It has now been found that the combination of antigenic regions ofToxoplasma gondii proteins, in the form of recombinant fusion products,retains the antigenic properties of the individual antigen fragments.The corresponding chimeric proteins thus produced can be used fordiagnostic and therapeutic purposes.

The use of said chimeric antigens as diagnostic agents and the relateddiagnostic aids containing them, for example in the form ofenzyme-linked immunoassays or kits, constitute a further object of thepresent invention.

Another object of the present invention are the gene sequences codingfor the above-mentioned chimeric antigens, their use as medicaments,particularly for the prevention and therapy of Toxoplasma gondiiinfection, e.g. as gene therapy. The present invention also extends tothe gene sequences that hybridize with the sequences of theabove-mentioned chimeric antigens in stringent hybridization conditions.

Another object of the present invention is the use of the chimericantigens as medicaments, particularly in the form of vaccines, which areuseful for the prevention and cure of the infection. The vaccinesaccording to the present invention are suitable for use in humans andother animals (particularly pig, cat, sheep).

These and other objects will be illustrated here below in detail, alsoby means of examples and figures.

DETAILED DESCRIPTION OF THE INVENTION

The main object of the present invention is, therefore, the provision ofrecombinant chimeric antigens obtained through the fusion of differentantigenic regions of Toxoplasma gondii gene products, and the use of therecombinant proteins thus obtained for developing selective diagnosticand therapeutic means.

The main advantages of the present invention over the other types ofantigens or antigen fragments known in the literature as reported aboveare the following and are evident when these antigens are used indiagnostic immunoassays on sample sera for detection of the infection:

-   -   With respect to the use of the entire Toxoplasma gondii antigen,        prepared from the parasite as lysed, whole-cell extract, the        chimeric recombinant antigens have the advantage of avoiding        unspecific reactions due to the presence of other        non-proteinaceous material and of providing a better        reproducibility. Moreover, some natural protein antigens of the        parasite are insoluble and, consequently, are poorly represented        in commercial assays employing the lysed, whole-cell extract        of T. gondii.    -   With respect to the use of single antigenic regions or single        antigen fragments (as described in WO 03/080839), the        recombinant chimeric antigens show the advantage of improving        the sensitivity of the assays in which they are used. In other        words their use decreases or abolishes the occurrence of false        negative responses.    -   A) With respect to the use of a mixture or a collection of        single antigenic regions (as also envisaged in WO 03/080839),        the advantages are least two. From the point of view of the        industrial applicability and production is much easier to        produce a single engineered construct containing three or more        antigen regions rather than separately produce each single        fragment and subsequently assemble them by an economic and        reproducible method. Secondly, as already said before, the use        of the chimeric recombinant antigens of the invention improves        the sensitivity of the assays.

These and other advantages are shown in the Examples section.

In particular the present invention relates to a chimeric recombinantantigen containing the fusion at least three different antigenic regionsof Toxoplasma gondii, wherein said antigenic regions are B-cellepitopes, which bind to Toxoplasma gondii-specific antibodies.Preferably the Toxoplasma gondii-specific antibodies are extracted fromsera of subjects who have been infected by Toxoplasma gondii.

More particularly the present invention covers a chimeric antigen,wherein the three different antigenic regions have an amino acidsequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.Preferred sequences in the above group are SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

For example the chimeric antigen of the invention comprises the aminoacid sequence of SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO:30 or the amino acid sequence of SEQ ID NO: 32.

The chimeric antigens of the present invention may be engineered usingknown methods. The fusions may be direct (the C-terminus of one aminoacid sequence is linked to the N-terminal of the other through a simplecovalent bond) or they may employ a flexible linker domain, such as thehinge region of human IgG, or polypeptide linkers consisting of smallamino acids such as glycine, serine, threonine or alanine, at variouslengths and combinations. For example the linker may be a polyglycinerepeat interrupted by serine or threonine at a certain interval.Preferably, the linker is composed by three glycine residues and twoserine residues, giving the aminoacid sequence Ser-Gly-Gly-Gly-Ser(SGGGS linker).

Additionally, the chimeric antigens of the invention may be tagged byHis-His-His-His-His-His (His6), to allow rapid purification bymetal-chelate chromatography, and/or by epitopes to which antibodies areavailable, to allow for detection on western blots, immunoprecipitation,or activity depletion/blocking in bioassays.

Another object of the present invention is a nucleotide sequence codingfor the chimeric antigen as defined above. According to a preferredembodiment of the invention such nucleotide sequence comprises at leastthree different nucleotide sequences selected from the group consistingof: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9and SEQ ID NO: 11. According to a more preferred embodiment, suchnucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 27or the nucleotide sequence of SEQ ID NO: 29 or the nucleotide sequenceof SEQ ID NO: 31.

Also included in the scope of the present invention are nucleotidesequences that hybridizes with any sequence described above understringent hybridization conditions, as well the corresponding chimericrecombinant antigen encoded by such hybridized nucleotide sequence.

The chimeric antigens of the present invention may be prepared bycloning and expression in a prokaryotic or eukaryotic expression system,using the appropriate expression vectors. Any method known in the artcan be employed.

For example the DNA molecules coding for the antigens of the inventionare inserted into appropriately constructed expression vectors bytechniques well known in the art (see Sambrook et al, 1989). Suchvectors are another object of the present invention.

In order to be capable of expressing the desired protein (in this casethe chimeric antigens), an expression vector should comprise alsospecific nucleotide sequences containing transcriptional andtranslational regulatory information linked to the DNA coding thedesired protein in such a way as to permit gene expression andproduction of the protein. First in order for the gene to betranscribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters).

For eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived form viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene, which has a high level ofexpression. Examples are the TK promoter of the Herpes virus, the SV40early promoter, the yeast gal4 gene promoter, etc. Transcriptionalinitiation regulatory signals may be selected which allow for repressionand activation, so that expression of the genes can be modulated. Allthese hosts are a further object of the present invention.

Nucleic acid molecules which encode the chimeric antigens of theinvention may be ligated to a heterologous sequence so that the combinednucleic acid molecule encodes a fusion protein. Such combined nucleicacid molecules are included within the embodiments of the invention. Forexample, they may be joined to the DNA coding for a protein which allowspurification of the chimeric antigen by only one step of affinitychromatography. This joined/fused protein may be for example GlutathioneSulpho Transferase (GST) to generate fusion products at the carboxyterminus of GST protein. The corresponding recombinant proteinsexpressed in the cytoplasm of transformed E. coli cells may be purifiedby affinity chromatography using a Glutathione-Sepharose resin.

The DNA molecule comprising the nucleotide sequence coding for thechimeric molecule of the invention is inserted into vector(s), havingthe operably linked transcriptional and translational regulatorysignals, which is capable of integrating the desired gene sequences intothe host cell. The cells which have been stably transformed by theintroduced DNA can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may also provide for phototrophy to an auxotrophichost, biocide resistance, e.g. antibiotics, or heavy metals such ascopper, or the like. The selectable marker gene can either be directlylinked to the DNA gene sequences to be expressed, or introduced into thesame cell by co-transfection. Additional elements may also be needed foroptimal synthesis of proteins of the invention.

Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells, that contain the vectormay be recognized and selected form those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Once the vector(s) or DNA sequence containing the construct(s) has beenprepared for expression the DNA construct(s) mat be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells may be either prokaryotic or eukaryotic. Example ofeukaryotic hosts are mammalian cells, such as human, monkey, mouse, andChinese hamster ovary (CHO) cells. Expression in these host cellsprovides post-translational modifications to protein molecules,including correct folding or glycosylation at correct sites. Also yeastcells can carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene products andsecretes peptides bearing leader sequences (i.e., pre-peptides). Exampleof prokaryotic hosts are bacteria, such as Escherichia coli.

After the introduction of the vector(s), the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired proteins.

Purification of the recombinant antigens is carried out by any one ofthe methods known for this purpose, i.e. any conventional procedureinvolving extraction, precipitation, chromatography, electrophoresis, orthe like. A further purification procedure that may be used inpreference for purifying the antigens of the invention is affinitychromatography using monoclonal antibodies which bind the target proteinand which are produced and immobilized on a gel matrix contained withina column. Impure preparations containing the recombinant protein arepassed through the column. The antigens will be bound to the column bythe specific antibody while the impurities will pass through. Afterwashing, the antigen is eluted from the gel by a change in pH or ionicstrength.

Another aspect of the present invention is the process for therecombinant production of the chimeric antigen as described above,comprising culturing the host cell transformed with the vectorcontaining the nucleotide sequence of the invention and isolating thedesired product.

A further object of the present invention is a DNA molecule comprisingthe DNA sequence coding for the above fusion protein, as well asnucleotide sequences substantially the same.

“Nucleotide sequences substantially the same” includes all other nucleicacid sequences which, by virtue of the degeneracy of the genetic code,also code for the given amino acid sequence.

Another object of the present invention is a nucleotide sequence whichhybridizes to the complement of the nucleotide sequence coding for thechimeric antigen of the invention under highly stringent or moderatelystringent conditions, as long as the antigen obtained maintains the samebiological activity, i.e. ability to bind to antibodies against theparasite.

The term “hybridization” as used here refers to the association of twonucleic acid molecules with one another by hydrogen bonding. Typically,one molecule will be fixed to a solid support and the other will be freein solution. Then, the two molecules may be placed in contact with oneanother under conditions that favour hydrogen bonding. Factors thataffect this bonding include: the type and volume of solvent; reactiontemperature; time of hybridization; agitation; agents to block thenon-specific attachment of the liquid phase molecule to the solidsupport (Denhardt's reagent or BLOTTO); the concentration of themolecules; use of compounds to increase the rate of association ofmolecules (dextran sulphate or polyethyleneglycol); and the stringencyof the washing conditions following hybridization.

Stringency conditions are a function of the temperature used in thehybridization experiment, the molarity of the monovalent cations and thepercentage of formamide in the hybridization solution. To determine thedegree of stringency involved with any given set of conditions, onefirst uses the equation of Meinkoth et al. (1984) for determining thestability of hybrids of 100% identity expressed as melting temperatureTm of the DNA-DNA hybrid: Tm=81.5° C.+16.6 (LogM)+0.41 (% GC)−0.61 (%form)−500/L, where M is the molarity of monovalent cations, % GC is thepercentage of G and C nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. For each 1° C. that the Tm is reduced from thatcalculated for a 100% identity hybrid, the amount of mismatch permittedis increased by about 1%. Thus, if the Tm used for any givenhybridization experiment at the specified salt and formamideconcentrations is 10° C. below the Tm calculated for a 100% hybridaccording to equation of Meinkoth, hybridization will occur even ifthere is up to about 10% mismatch.

As used herein, highly stringent conditions are those which are tolerantof up to about 15% sequence divergence, while moderately stringentconditions are those which are tolerant of up to about 20% sequencedivergence. Without limitation, examples of highly stringent (12-15° C.below the calculated Tm of the hybrid) and moderately (15-20° C. belowthe calculated Tm of the hybrid) conditions use a wash solution of 2×SSC(standard saline citrate) and 0.5% SDS at the appropriate temperaturebelow the calculated Tm of the hybrid. The ultimate stringency of theconditions is primarily due to the washing conditions, particularly ifthe hybridization conditions used are those which allow less stablehybrids to form along with stable hybrids. The wash conditions at higherstringency then remove the less stable hybrids. A common hybridizationcondition that can be used with the highly stringent to moderatelystringent wash conditions described above is hybridization in a solutionof 6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA at a temperature approximately20° C. to 25° C. below the Tm. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC(Ausubel, 1987-1998).

The term “nucleic acid molecule” also includes analogues of DNA and RNA,such as those containing modified backbones.

The nucleic acid molecules of the invention also include antisensemolecules that are partially complementary to nucleic acid moleculesencoding antigens of the present invention and that therefore hybridizeto the encoding nucleic acid molecules (hybridization). Such antisensemolecules, such as oligonucleotides, can be designed to recognise,specifically bind to and prevent transcription of a target nucleic acidencoding a polypeptide of the invention, as will be known by those ofordinary skill in the art (see, for example, Cohen, J. S., Trends inPharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991);O'Connor, J. Neurochem 56, 560 (1991); Lee et al., Nucleic Acids Res 6,3073 (1979); Cooney et al., Science 241, 456 (1988); Dervan et al.,Science 251, 1360 (1991).

According to the terminology used herein, a composition containing acompound [X] is “substantially free of” impurities [herein, Y] when atleast 85% by weight of the total X+Y in the composition is X.Preferably, X comprises at least about 90% by weight of the total of X+Yin the composition, more preferably at least about 95%, 98% or even 99%by weight.

Another aspect of the invention is the use of chimeric antigensdescribed above as medicaments. In particular, one of the main objectsof the invention is use of chimeric antigens as active ingredients forthe preparation of medicaments for the prevention or treatment ofToxoplasma gondii infections.

In the case of gene therapy another object of the invention is the useof the nucleotide sequences coding for the antigens of the invention asmedicaments, in particular for the preparation of medicaments useful forthe treatment and prevention of Toxoplasma gondii infections.

The pharmaceutical compositions should preferably comprise atherapeutically effective amount of the chimeric antigens of theinvention or the corresponding nucleotide sequence. Chimeric antigens ofthe invention may thus act as vaccines for the prevention or thetreatment of Toxoplasma gondii infection.

For the therapeutic application, where the preparation of medicaments orvaccines comes within the framework of general knowledge for furtherreference the reader is again referred to the patent literature cited inthe present description and, particularly, to Beghetto et al, TheJournal of Infectious Diseases, 2005, 191:637-645.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent needed to treat, ameliorate, or prevent atargeted disease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any compound, the therapeutically effectivedose can be estimated initially either in cell culture assays, forexample, of neoplastic cells, or in animal models, usually mice,rabbits, dogs, or pigs.

The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

The precise effective amount for a human subject will depend upon theseverity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination (s), reaction sensitivities, andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05mg/kg to 10 mg/kg. Compositions may be administered individually to apatient or may be administered in combination with other agents, drugsor hormones.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier, for administration of a therapeutic agent. Suchcarriers include antibodies and other polypeptides, genes and othertherapeutic agents such as liposomes, provided that the carrier does notitself induce the production of antibodies harmful to the individualreceiving the composition, and which may be administered without unduetoxicity.

Suitable carriers may be large, slowly metabolised macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulphates, and the like; and the salts of organic acids such asacetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable carriers is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such compositions. Such carriers enable the pharmaceuticalcompositions to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, foringestion by the patient.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal or transcutaneousapplications (for example, see W098/20734), subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, intravaginalor rectal means. Gene guns or hyposprays may also be used to administerthe pharmaceutical compositions of the invention. Typically, thetherapeutic compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion.

Dosage treatment may be a single dose schedule or a multiple doseschedule.

The method of treating a mammal suffering from Toxoplasma-gondiiinfection, comprising administering a therapeutically effective amountof the vaccine as described above represents one of the aspects of thepresent invention.

A further object of the present invention is the use of chimericantigens as described above as active agents for the diagnosis ofToxoplasma gondii infections, in particular for the diagnosis of thetime of infection.

Also the kits for the diagnosis of Toxoplasma gondii infection,containing at least one chimeric antigen according are part of thepresent invention. Such kits may be useful for the diagnosis of an acuteand/or chronic Toxoplasma gondii infection.

The chimeric antigen of the invention may be employed in virtually anyassay format that employs a known antigen to detect antibodies. A commonfeature of all of these assays is that the antigen is contacted with thebody component suspected of containing antibodies under conditions thatpermit the antigen to bind to any such antibody present in thecomponent. Such conditions will typically be physiologic temperature, pHand ionic strength using an excess of antigen. The incubation of theantigen with the specimen is followed by detection of immune complexescomprised of the antigen.

Design of the immunoassays is subject to a great deal of variation, andmany formats are known in the art. Protocols may, for example, use solidsupports, or immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the immune complex are also known;examples of which are assays which utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

The immunoassay may be, without limitation, in a heterogenous or in ahomogeneous format, and of a standard or competitive type. In aheterogeneous format, the polypeptide is typically bound to a solidmatrix or support to facilitate separation of the sample from thepolypeptide after incubation.

Examples of solid supports that can be used are nitrocellulose (e.g., inmembrane or microtiter well form), polyvinyl chloride (e.g., in sheetsor microtiter wells), polystyrene latex (e.g., in beads or microtiterplates, polyvinylidine fluoride (known as Immulon™), diazotized paper,nylon membranes, activated beads, and Protein A beads. For example,Dynatech Immulon™1 or Immulon™2 microtiter plates or 0.25 inchpolystyrene beads (Precision Plastic Ball) can be used in theheterogeneous format. The solid support containing the antigenicpolypeptides is typically washed after separating it from the testsample, and prior to detection of bound antibodies. Both standard andcompetitive formats are known in the art.

In a homogeneous format, the test sample is incubated with thecombination of antigens in solution. For example, it may be underconditions that will precipitate any antigen-antibody complexes whichare formed. Both standard and competitive formats for these assays areknown in the art.

In a standard format, the amount of antibodies forming theantibody-antigen complex is directly monitored. This may be accomplishedby determining whether labeled anti-xenogenic (e.g., anti-human)antibodies which recognize an epitope on anti-Toxoplasma gondiiantibodies will bind due to complex formation. In a competitive format,the amount of antibodies in the sample is deduced by monitoring thecompetitive effect on the binding of a known amount of labeled antibody(or other competing ligand) in the complex.

Complexes formed comprising anti-Toxoplasma gondii antibody (or, in thecase of competitive assays, the amount of competing antibody) aredetected by any of a number of known techniques, depending on theformat. For example, unlabeled antibodies in the complex may be detectedusing a conjugate of antixenogeneic Ig complexed with a label, (e.g., anenzyme label).

In an immunoprecipitation or agglutination assay format the reactionbetween the chimeric antigens and the antibody forms a network thatprecipitates from the solution or suspension and forms a visible layeror film of precipitate. If no anti-Toxoplasma gondii antibody is presentin the test specimen, no visible precipitate is formed.

The chimeric antigens of the invention will typically be packaged in theform of a kit for use in these immunoassays. The kit will normallycontain in separate containers the combination of antigens (eitheralready bound to a solid matrix or separate with reagents for bindingthem to the matrix), control antibody formulations (positive and/ornegative), labeled antibody when the assay format requires same andsignal generating reagents (e.g., enzyme substrate) if the label doesnot generate a signal directly. Instructions (e.g., written, tape, VCR,CD-ROM, etc.) for carrying out the assay usually will be included in thekit.

The diagnostic kits, which are the object of the present invention, aretherefore known to the expert in the field. By way of an example, thereader is referred to the patent literature cited above, to which may beadded U.S. Pat. No. 6,265,176, WO 01/63283, and WO 03/080839 as furtherreferences.

The invention will now be illustrated in greater detail by means ofexamples and figures.

DESCRIPTION OF THE FIGURES

FIG. 1. Plasmid map of the bacterial expression vector pGEX-SN.

FIG. 2. Schematic representation of the chimeric antigens.

The DNA sequences of clones Tx-2.a, Tx-1.16, Tx-4.18, Tx-15.11, Tx-1.11and Tx-11.b, respectively encoding for protein fragments of the T.gondii genes MIC2, MIC3, SAG1, GRA3, GRA7 and M2AP were used for theconstruction of GST-EC2, GST-EC3 and GST-EC4 fusion proteins.

FIG. 3. Expression of T. gondii chimeric antigens in E. coli cells.

SDS-PAGE analysis of purified GST-EC2, GST-EC3 and GST-EC4 fusionproteins. The recombinant proteins were subjected to electrophoresis(0.003 mg/lane) on 12% acrylamide gel. MW, molecular weight markers.

FIG. 4. Biochemical characterisation of the chimeric antigens

HPLC analysis of the purified GST-EC2 performed by gel filtration usingthe TSK G4000 SW-XL column.

FIG. 5. Antigenic properties of individual protein fragments within thechimeric antigens.

Immunoreactivity of individual Tx-2.a, Tx-1.16, Tx-4.18, Tx-15.11,Tx-1.11 and Tx-11.b antigen fragments, and of EC2, EC3 and EC4 chimericantigens with serum samples from T. gondii infected individuals. Serawere used either as whole specimens (serum) or after depletion ofspecific antibodies against combinations of antigen fragments(Tx-1.16/Tx-4.18 depletion, Tx-2.a/Tx-4.18 depletion, etc.).

FIG. 6. Immunoreactivity of the chimeric antigens withToxoplasma-specific IgM antibodies

-   A) IgM Rec-ELISA analysis performed with Toxoplasma-specific    IgM-positive (C+) and IgM-negative (C−) serum samples (sera    dilution, 1:100) by varying the concentration of the biotin-labeled    GST-EC2 and GST-EC3 antigens.-   B) IgM Rec-ELISA analysis performed with serial dilutions of    Toxoplasma-specific IgM-positive (from 1:100 to 1:6400) and    IgM-negative (the control serum, diluted 1:100) serum samples,    assayed with the biotin-labeled GST-EC2 and the mixture of    GST-EC2/GST-EC3.

FIG. 7. Long-term stability of the chimeric antigens

IgM Rec-ELISA analysis performed with the biotin-labeled GST-EC2 andGST-EC3 antigens, which have been maintained at +4° C. for differenttime intervals (days). Results are expressed as the ratio of the OpticalDensities measured with the Toxoplasma-specific IgM-positive (OD C+) andIgM-negative (OD C−) serum samples.

EXAMPLES

The following Table 1 gives, by way of examples, the DNA sequences usedfor the construction of recombinant Toxoplasma gondii chimeric antigens:

TABLE 1 Name Sequence Identification Classification Tx-15.11GCTGCCTTGGGAGGCCTTGCGGCGGATC GRA 3 Dense (SEQ ID 1)AGCCTGAAAATCATCAGGCTCTTGCAGAA granule CCAGTTACGGGTGTGGGGGAAGCAGGAprotein GTGTCCCCCGTCAACGAAGCTGGTGAGT CATACAGTTCTGCAACTTCGGGTGTCCAAGAAGCTACCGCCCCAGGTGCAGTGCTCC TGGACGCAATCGATGCCGAGTCGGATAAGGTGGACAATCAGGCGGAGGGAGGTGA GCGTATGAAGAAGGTCGAAGAGGAGTTGTCGTTATTGAGGCGGGAATTATATGATCG CACAGATCGCCCTGGT Tx-1.11CAGTTCGCTACCGCGGCCACCGCGTCAG GRA 7 Dense (SEQ ID 3)ATGACGAACTGATGAGTCGAATCCGAAAT granule TCTGACTTTTTCGATGGTCAAGCACCCGTprotein TGACAGTCTCAGACCGACGAACGCCGGT GTCGACTCGAAAGGGACCGACGATCACCTCACCACCAGCATGGATAAGGCATCTGTA GAGAGTCAGCTTCCGAGAAGAGAGCCATTGGAGACGGAGCCAGATGAACAAGAAGA AGTTCAT Tx-1.16AGGAGGACTGGATGTCATGCCTTCAGGG MIC 3 Microneme (SEQ ID 5)AGAACTGCAGCCCTGGTAGATGTATTGAT protein GACGCCTCGCATGAGAATGGCTACACCTGCGAGTGCCCCACAGGGTACTCACGTGA GGTGACTTCCAAGGCGGAGGAGTCGTGTGTGGAAGGAGTCGAAGTCACGCTGGCTG AGAAATGCGAGAAGGAATTCGGCATCAGCGCGTCATCCTGCAAATGCGAT Tx-4.18 CCATCGGTCGTCAATAATGTCGCAAGGT SAG 1Surface (SEQ ID 7) GCTCCTACGGTGCAGACAGCACTCTTGG proteinTCCTGTCAAGTTGTCTGCGGAAGGACCC ACTACAATGACCCTCGTGTGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACAACAAT CAGTACTGTTCCGGGACGACGCTGACTGGTTGCAACGAGAAATCGTTCAAAGATATT TTGCCAAAATTAACTGAGAACCCGTGGCAGGGTAACGCTTCGAGTGATAAGGGTGCC ACGCTAACGATCAAGAAGGAAGCATTTCCAGCCGAGTCAAAAAGCGTCATTATTGGAT GCACAGGGGGATCGCCTGAGAAGCATCACTGTACCGTGAAACTGGAGTTTGCCGGG GCTGCAGGGTCAGCAAAATCGGCT Tx-2.aCCCCAGGATGCCATTTGCTCGGATTGGT MIC2 Microneme (SEQ ID 9)CCGCATGGAGCCCCTGCAGTGTATCCTG protein CGGTGACGGAAGCCAAATCAGGACGCGAACTGAGGTTTCTGCTCCGCAACCTGGAA CACCAACATGTCCGGACTGCCCTGCGCCCATGGGAAGGACTTGCGTGGAACAAGGC GGACTTGAAGAAATCCGTGAATGCAGTGCGGGGGTATGTGCTGTTGACGCTGGATG TGGCGTCTGGGTT Tx-11.bAACGAACCGGTGGCCCTAGCTCAGCTCA M2AP Microneme (SEQ ID 11)GCACATTCCTCGAGCTCGTCGAGGTGCC protein ATGTAACTCTGTTCATGTTCAGGGGGTGATGACCCCGAATCAAATGGTCAAAGTGACT GGTGCAGGATGGGATAATGGCGTTCTCGAGTTCTATGTCACGAGGCCAACGAAGAC AGGCGGGGACACAAGCCGAAGCCATCTTGCGTCGATCATGTGTTATTCCAAGGACAT TGACGGCGTGCCGTCAGACAAAGCGGGAAAGTGCTTTCTGAAGAACTTTTCTGGTGA AGACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACCCATCAAGAGCCACAACGA TGCGTTCATGTTCGTTTGTTCTTCAAATGATGGATCCGCACTCCAGTGTGATGTTTTCG CCCTTGATAACACCAACTCTAGCGACGGGTGGAAAGTGAATACCGTGGATCTTGGC GTCAGCGTTAGTCCGGATTTGGCATTCGGACTCACTGCAGATGGGGTCAAGGTGAA GAAGTTGTACGCAAGCAGCGGCCTGACAGCGATCAACGACGACCCTTCCTTGGGGT GCAAGGCTCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGTTTGCCGTCGCCT GAAAACAGCGGGTCTGCAACACCAGCGGAAGAAAGTCCGTCTGAGTCTGAATCT

The sequence Tx-15.11 constitutes a fragment of the gene GRA3 (Bermudeset al., Mol. Biochem. Parasitol., 1994, 68:247-257). Said clone has theamino acid sequence AALGGLAADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQEATAPGAVLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRELYDRTDRPG (SEQ ID 2) and its usein chimeric antigens is covered by the present invention.

The sequence Tx-1.11 constitutes a fragment of the antigen GRA7(Bonhomme et al., J. Histochem. Cytochem., 1998, 46:1411-1421). Saidclone has the amino acid sequenceATAATASDDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDKASVESQLPRREPLETEPDEQEEVHF (SEQ ID 4) and its use in chimeric antigens iscovered by the present invention.

The sequence Tx-1.16 constitutes a fragment of the MIC3 gene(Garcia-Réguet et al., Cellular Microbiol., 2000, 2:353-364). Said clonehas the amino acid sequenceRRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCD (SEQ ID 6) and its use in chimeric antigens iscovered by the present invention.

The sequence Tx-4.18 constitutes a fragment of the antigen SAG1 (Burg etal., J. Immunol., 1988, 141:3584-3591). Said clone has the amino acidsequence PSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESKSVIIGCTGGSPEKHHCTVKLEFAGAAGSAKSA (SEQ ID 8) and its use in chimeric antigensis covered by the present invention.

The sequence Tx-2.a represents a fragment of the MIC2 gene (Wan et al,Mol. Biochem. Parasitol., 1997, 84:203-214). Said clone has the aminoacid sequence PQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTCVEQGGLEEIRECSAGVCAVDAGCGVWV (SEQ ID 10) and its use in chimericantigens is covered by the present invention.

The sequence Tx-11.b represents a distinct fragment of the M2AP gene(Rabenau et al., Mol. Microbiol., 2001, 41:537-547). Said clone has theamino acid sequence NEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVSVSPDLAFGLTADGVKVKKLYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPE NSGSATPAEESPSESES(SEQ ID 12) and its use in chimeric antigens is covered by the presentinvention.

Construction of Chimeric Antigens

EC2 protein product is a chimeric molecule containing the DNA sequencesTx-2.a, Tx-1.16 and Tx-4.18.

SEQ ID 9 was used as template for DNA amplification of clone Tx-2.a byusing oligonucleotides K551 (5′-GGACTAGTCGGCTCCCCCAGGATGCC-3′) and K553(5′-CATCCAGTCCTGCTACCGCCACCAGACCAGACGCCACATCC AGC-3′). Theoligonucleotide K553 contains a sequence encoding for the linker SGGGS,which joins the sequences Tx-2.a and Tx-1.16. PCR condition was 30″ at94° C., 30″ at 50° C. and 60″ at 72° C. for 20 cycles.

SEQ ID 5 was used as template for DNA of clone Tx-1.16 by usingoligonucleotides K552 (5′-GTGGCGTCTGGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCC-3′) and K555 (5′-TGACGACCGAGCTACCGCCACCAGAGTTATCGCATTTTGCAGGATG-3′). The oligonucleotide K555 contains asequence encoding for the linker SGGGS, which joins the sequencesTx-1.16 and Tx-4.18. PCR condition was 30″ at 94° C., 30″ at 50° C. and60″ at 72° C. for 20 cycles.

SEQ ID 7 was used as template for DNA amplification of clone Tx-4.18 byusing oligonucleotides K554 (5′-ATGCGATAACTCTGGTGGCGGTAGCTCGGTCGTCAATAATGTCGC-3′) and K556 (5′-CCGCGGCCGCTAGCCGATTTTGCTGACCCTG-3′). PCR condition was 30″ at 94° C., 30″ at 50°C. and 60″ at 72° C. for 20 cycles.

The PCR products were purified by means of the “Qiagen Purification Kit”(Qiagen, CA, USA). 25 ng of DNA amplification products of SEQ ID 9 andSEQ ID 5 were mixed together and used as templates in PCR reaction byusing oligonucleotides K551 and K555. PCR condition was 30″ at 94° C.,30″ at 50° C. and 60″ at 72° C. for 20 cycles. 25 ng of the resultingDNA amplification was purified with “Qiagen Purification Kit” (Qiagen,CA, USA) and then mixed with 25 ng of DNA amplification product of SEQID 4. Finally, the DNA mixture was used as template for DNAamplification by using oligonucleotides K551 and K556, following PCRcondition of 30″ at 94° C., 30″ at 50° C. and 90″ at 72° C. for 20cycles.

EC3 protein product is a chimeric molecule containing the DNA sequencesTx-15.11, Tx-1.11 and Tx-11.b.

SEQ ID 1 was used as template for DNA amplification of clone Tx-15.11 byusing oligonucleotides K563 (5′-GGACTAGTCGGCTGG CTGCCTTGGGAGGCCTTG-3′)and K565 (5′-GCCGCGGTAGCACTACCG CCACCAGACAAACCAGGGCGATCTGTG-3′). Theoligonucleotide K565 contains a sequence encoding for the linker SGGGS,which joins the sequences Tx-15.11 and Tx-1.11. The PCR protocol was 30″at 94° C., 30″ at 48° C. and 60″ at 72° C. for 20 cycles.

SEQ ID 3 was used as template for DNA amplification of clone Tx-1.11 byusing oligonucleotides K564 (5′-GCCCTGGTTTGTCTGGTGGCGGTAGTGCTACCGCGGCCACCGCG-3′) and K567 (5′-CCGGTTCGTTACTACCGCCACCAGAGAAATGAACTTCTTCTTGTTC-3′). The oligonucleotide K567 contains asequence encoding for the linker SGGGS, which joins the sequencesTx-1.11 and Tx-11.b. The PCR protocol was 30″ at 94° C., 30″ at 48° C.and 60″ at 72° C. for 20 cycles.

SEQ ID 11 was used as template for DNA amplification of clone Tx-11.b byusing oligonucleotides K566 (5′-GAAGTTCATTTCTCTGGTGGCGGTAGTAACGAACCGGTGGCCCTAG-3′) and K568 (5′-CCGCGGCCGCAGATTCAGACTCAGACGGAC-3′). The PCR protocol was 30″ at 94° C., 30″ at 45°C. and 60″ at 72° C. for 20 cycles.

The PCR products were purified by means of the “Qiagen Purification Kit”(Qiagen, CA, USA). 25 ng of DNA amplification products of SEQ ID 1 andSEQ ID 3 were mixed together and used as templates in PCR reaction byusing oligonucleotides K563 and K567. The PCR protocol was 30″ at 94°C., 30″ at 45° C. and 60″ at 72° C. for 30 cycles. 25 ng of theresulting DNA amplification was purified and then mixed with 25 ng ofDNA amplification product of SEQ ID 11. Finally, the DNA mixture wasused as template for DNA amplification by using oligonucleotides K563and K568, following PCR condition of 30″ at 94° C., 30″ at 45° C. and180″ at 72° C. for 30 cycles.

EC4 protein product is a chimeric molecule containing the DNA sequencesTx-2.a, Tx-1.16 and Tx-11.b.

SEQ ID 9 was used as template for DNA amplification of clone Tx-2.ausing oligonucleotides K551 and K553.

SEQ ID 5 was used as template for DNA amplification of clone Tx-1.16 byusing oligonucleotides K552 and K572 (5′-CGTTACTACCGCCACCAGAGTTATCGCATTTGCAGGATGA-3′). The oligonucleotide K572 contains asequence encoding for the linker SGGGS, which joins the sequencesTx-1.16 and Tx-11.b.

SEQ ID 11 was used as template for DNA amplification of clone Tx-11.b byusing oligonucleotides K571 (5′-TAACTCTGGTGGCGGTAGTAACGAACCGGTGGCCCTAGC-3′) and K568.

The PCR products were purified as by using “Qiagen Purification Kit”(Qiagen, CA, USA). 25 ng of DNA amplification products of SEQ ID 9 andSEQ ID 5 were mixed together and used as templates in PCR reaction byusing oligonucleotides K551 and K572. 25 ng of the resulting DNAamplification was purified and then mixed with 25 ng of DNAamplification product of SEQ ID 11. Finally, the DNA mixture was used astemplate for DNA amplification by using oligonucleotides K551 and K568.PCR conditions for the construction of EC4 were the same that those usedfor EC2 and EC3 constructs.

The following Table 2 gives, by way of examples, the DNA sequences ofthe EC2, EC3 and EC4 chimeric antigens:

TABLE 2 Name Sequence EC2 ACTAGTCGGCTCCCCCAGGATGCCATTTGCTCGGATTGGTC SEQCGCATGGAGCCCCTGCAGTGTATCCTGCGGTGACGGAAGC ID 27CAAATCAGGACGCGAACTGAGGTTTCTGCTCCGCAACCTGGAACACCAACATGTCCGGACTGCCCTGCGCCCATGGGAAGGACTTGCGTGGAACAAGGCGGACTTGAAGAAATCCGTGAATGCAGTGCGGGGGTATGTGCTGTTGACGCTGGATGTGGCGTCTGGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCCTTCAGGGAGAACTGCAGCCCTGGTAGATGTATTGATGACGCCTCGCATGAGAATGGCTACACCTGCGAGTGCCCCACAGGGTACTCACGTGAGGTGACTTCCAAGGCGGAGGAGTCGTGTGTGGAAGGAGTCGAAGTCACGCTGGCTGAGAAATGCGAGAAGGAATTCGGCATCAGCGCGTCATCCTGCAAATGCGATAACTCTGGTGGCGGTAGCTCGGTCGTCAATAATGTCGCAAGGTGCTCCTACGGTGCAGACAGCACTCTTGGTCCTGTCAAGTTGTCTGCGGAAGGACCCACTACAATGACCCTCGTGTGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACAACAATCAGTACTGTTCCGGGACGACGCTGACTGGTTGCAACGAGAAATCGTTCAAAGATATTTTGCCAAAATTAACTGAGAACCCGTGGCAGGGTAACGCTTCGAGTGATAAGGGTGCCACGCTAACGATCAAGAAGGAAGCATTTCCAGCCGAGTGAAAAAGCGTCATTATTGGATGCACAGGGGGATCGCCTGAGAAGCATCACTGTACCGTGAAACTGGAGTTTGCCGGGGCTGCAGGGTCAGCAAAATCGGCTAGCGGCCGC EC3ACTAGTCGGCTGGCTGCCTTGGGAGGCCTTGCGGATCAGC SEQCTGAAAATCATCAGGCTCTTGCAGAACCAGTTACGGGTGTG ID 29GGGGAAGCAGGAGTGTCCCCCGTCAACGAAGCTGGTGAGTCATACAGTTCTGCAACTTCGGGTGTCCAAGAAGCTACCGCCCCAGGTGCAGTGCTCCTGGACGCAATCGATGCCGAGTCGGATAAGGTGGACAATCAGGCGGAGGGAGGTGAGCGTATGAAGAAGGTCGAAGAGGAGTTGTCGTTATTGAGGCGGGAATTATATGATCGCACAGATCGCCCTGGTTTGTCTGGTGGCGGTAGTGCTACCGCGGCCACCGCGTCAGATGACGAACTGATGAGTCGAATCCGAAATTCTGACTTTTTCGATGGTCAAGCACCCGTTGACAGTCTCAGACCGACGAACGCCGGTGTCGACTCGAAAGGGACCGACGATCACCTCACCACCAGCATGGATAAGGCATCTGTAGAGAGTCAGCTTCCGAGAAGAGAGCCATTGGAGACGGAGCCAGATGAACAAGAAGAAGTTCATTTCTCTGGTGGCGGTAGTAACGAACCGGTGGCCCTAGCTCAGCTCAGCACATTCCTCGAGCTCGTCGAGGTGCCATGTAACTCTGTTCATGTTCAGGGGGTGATGACCCCGAATCAAATGGTCAAAGTGACTGGTGCAGGATGGGATAATGGCGTTCTCGAGTTCTATGTCACGAGGCCAACGAAGACAGGCGGGGACACAAGCCGAAGCCATCTTGCGTCGATCATGTGTTATTCCAAGGACATTGACGGCGTGCCGTCAGACAAAGCGGGAAAGTGCTTTCTGAAGAACTTTTCTGGTGAAGACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACCCATCAAGAGCCACAACGATGCGTTCATGTTCGTTTGTTCTTCAAATGATGGATCCGCACTCCAGTGTGATGTTTTCGCCCTTGATAACACCAACTCTAGCGACGGGTGGAAAGTGAATACCGTGGATCTTGGCGTCAGCGTTAGTCCGGATTTGGCATTCGGACTCACTGCAGATGGGGTCAAGGTGAAGAAGTTGTACGCAAGCAGCGGCCTGACAGCGATCAACGACGACCCTTCCTTGGGGTGCAAGGCTCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGTTTGCCGTCGCCTGAAAACAGCGGGTCTGCAACACCAGCGGAAGAAAGTCCGTCTGAGTCTGAATCTGCGGCCGCGG EC4ACTAGTCGGCTCCCCCAGGATGCCATTTGCTCGGATTGGTC SEQCGCATGGAGCCCCTGCAGTGTATCCTGCGGTGACGGAAGC ID 31CAAATCAGGACGCGAACTGAGGTTTCTGCTCCGCAACCTGGAACACCAACATGTCCGGACTGCCCCGCGCCCATGGGAAGGACTTGCGTGGAACAAGGCGGACTTGAAGAAATCCGTGAATGCAGTGCGGGGGTATGTGCTGTTGACGCTGGATGTGGCGTCTGGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCCTTCAGGGAGAACTGCCGCCCTGGTAGATGTATTGATGACGCCTCGCATGAGAATGGCTACACCTGCGAGTGCCCCACATGGTACTCACGTGAGGTGACTTCCAAGGCGGAGGAGTCGTGTGTGGAAGGAGTCGAAGTCACGCTGGCTGAGAAATGCGAGAAGGAATTCGGCATCAGCGCGTCCTCCTGCAAATGCGATAACTCTGGTGGCGGTAGTAACGAACCGGTGGCCCTAGCTCAGCTCAGCACATTCCTCGAGCTCGTCGAGGTGCCATGTAACTCTGTTCATGTTCAGGGGGTGATGACCCCGAATCAAATGGTCAAAGTGACTGGTGCAGGATGGGATAATGGCGTTCTCGAGTTCTATGTCACGAGGCCAACGAAGACAGGCGGGGACACAAGCCGAAGCCACCTTGCGTCGATCATGTGTTATTCCAAGGACATTGACGGCGTGCCGTCAGACAAAGCGGGAAAGTGCTTTTTGAAGAACTTTTCTGGTGAAGACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACCCATCAAGAGCCACAACGATGCGTTCATGTTCGTTTGTTCTTCAAATGATGGATCCGCACTCCAGTGTGATGTTTTCGCCCTTGATAACACCAACTCTAGCGACGGGTGGAAAGTGAATACCGTGGATCTTGACGTCAGCGTTAGTCCGGATTTGGCATTCGGACTCACTGCAGATGGGGTCAAGGTGAAGAAGTTGTACGCAAGCAGCGGCCTGACAGCGATCAACGACGACCCTTCCTTGGGGTGCAAGGCTCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGTTTGCCGTCGCCTGAAAACAGCGGGTCTGCAACACCAGCGGAAGAAAGTCCGTCTGAGTCTGAATCTGCGGCCGCGG

The chimeric protein EC2 has the amino acid sequenceTSRLPQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTCVEQGGLEEIRECSAGVCAVDAGCGVWSGGGSRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCDNSGGGSSWNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESKSVIIGCTGGSPEKHHCTVKLEFAGMGSAKSASGR (SEQ ID 28) and its use asrecombinant antigen, containing multiple Toxoplasma gondii proteinfragments, is covered by the present invention.

The chimeric protein EC3 has the amino acid sequenceTSRLAALGGLADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQEATAPGAVLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRELYDRTDRPGLSGGGSATAATASDDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDKASVESQLPRREPLETEPDEQEEVHFSGGGSNEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVSVSPDLAFGLTADGVKVKKLYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENSGSATPAEESPSESES AAA (SEQ ID 30)and its use as recombinant antigen, containing multiple Toxoplasmagondii protein fragments, is covered by the present invention.

The chimeric protein EC4 has the amino acid sequenceTSRLPQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTCVEQGGLEEIRECSAGVCAVDAGCGVWSGGGSRTGCHAFRENCRPGRCIDDASHENGYTCECPTWYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCDNSGGGSNEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLDVSVSPDLAFGLTADGVKVKKLYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENSGSATPAEESPSESESAAA (SEQ ID 32) and its use as recombinantantigen, containing multiple Toxoplasma gondii protein fragments, iscovered by the present invention.

Construction of DNA Vectors Directing the Expression of ChimericAntigens as Fusion Products with GST in the Cytoplasm of E. coli Cells

DNA fragments encoding for the EC2, EC3 and EC4 chimeric proteins werecloned as fusion products with the protein Glutathione SulphoTransferase (GST) and expressed as soluble proteins in the cytoplasm ofbacterial cells, for the purpose of determining their specificity andselectivity. DNA sequences of EC2, EC3 and EC4 (SEQ ID 27, 29 and 31,respectively) were digested with the restriction enzymes SpeI and NotI.Digested DNA were cloned into vector pGEX-SN (Minenkova et al.,International Journal of Cancer, 2003, 106:534-44), which was previouslydigested with SpeI and NotI endonucleases, to generate fusion productsat the carboxy terminus of GST protein. The resulting plasmids were usedto transform competent E. coli cells following standard protocols(Sambrook et al., 1989, Molecular Cloning, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor).

Biochemical Characterisation of the Recombinant Chimeric Antigens

The recombinant proteins GST-EC2, GST-EC3 and GST-EC4 were expressed inthe cytoplasm of transformed E. coli cells and purified by affinitychromatography using Glutathione-Sepharose resin (Amersham PharmaciaBiotech, Sweden), following the manufacturer's instructions. Proteinpurity and concentration were assessed by SDS-PAGE (Sodium DodecylSulphate-Poly-acrylamide Gel Electrophoresis) analysis and Bradfordassay, respectively. The affinity-purified recombinant products weredialyzed against PBS, diluted at a concentration of 1 mg/ml with PBS andstored at −20° C. until use. The yield of purified products were 8mg/liter of bacterial culture, 5 mg/liter and 4 mg/liter for thechimeric antigens GST-EC2, GST-EC3 and GST-EC4, respectively. Therecombinant proteins were subsequently subjected to high-performanceliquid chromatography (HPLC) analysis. To this aim a gel filtration wasperformed using a TSK G4000 SW-XL HPLC-column, by injecting 30 μl ofeach samples (protein concentration, 2 mg/ml) into the column with aflow rate of 1 ml/min (mobile phase: KH₂PO₄ 0.05 M; NaCl 0.3 M pH 7.0).Results from HPLC analysis demonstrated that the chimeric antigensGST-EC2, GST-EC3 and GST-EC4 were purified as homogeneous products indimeric forms (see FIG. 4 were the HPLC analysis of GST-EC2 is shown asan example), with a respective molecular weight of 110.4, 152.6 and 130kDa (Da, Dalton). Protein aggregates of high molecular weight wereabsent in all of the purified protein preparations.

Immunoreactivity of the Chimeric Recombinant Antigens with IgGAntibodies from Sera of T. gondii Infected Individuals: IgG Rec-ELISA

The ELISA performance of the GST fusion products was performed bycoating Maxisorp-multiwells plates (Nunc) with single antigen fragmentsor chimeric proteins at a concentration of 5 μg/ml in coating buffer (50mM NaHCO₃, pH 9.6). After incubation overnight at 4° C. plates wereincubated for 1 h at 37° C. with blocking buffer (5% non-fat dry milk,0.05% Tween-20 in PBS) and subsequently incubated for 1 h at 37° C. withsera from T. gondii seropositive and seronegative individuals, diluted1:200 in blocking solution. Plates were extensively washed with 0.05%Tween-20 in PBS and anti-human-IgG horse-radish peroxidase-conjugatedantibodies (1 mg/ml; Sigma-Aldrich, USA), diluted 1:10000 in blockingsolution, was then added to each well. Finally, incubating plates withthe chromogenic substrate tetramethylbenzidine (TMB; Sigma-Aldrich, USA)revealed the enzymatic activity. Results were recorded as the differencebetween the absorbance (Optical Density, OD) at 450 and 620 nm, detectedby an automated ELISA reader (Labsystem Multiskan, Finland). For eachserum sample the assay was done in triplicate and average values werecalculated.

The following Table 3 shows the IgG reactivity of the single antigenfragments and EC2, EC3 and EC4 chimeric antigens, expressed as GSTfusion proteins, by using sera from 36 (T1-T36) T. gondii-seropositiveand 27 (N1-N27) T. gondii-seronegative humans. The Toxoplasma-specificIgG levels, calculated as International Units (IU) using a commercialassay (VIDAS system, bioMerieux, Marcy-l'Etoile, France) are reported.For every GST-fusion product the cut-off value was determined as themean plus two times the standard deviation of the absorbency readingsobtained from the Toxoplasma IgG negative sera. Normal type, OD<cut-off;bold type, OD>cut-off. The numerical value reported into each cell wascalculated as the ratio of the test OD to the cut-off of thecorresponding antigen (nd, not determined). Values greater than 1indicate a positive response.

Table 3 clearly shows that some of the sera which are positive result tobe negative when using single antigenic fragments, whereas they resultto be positive (correctly) when using the chimeric antigens of thepresent invention. Please note that the numerical values of IgGconcentrations obtained with the standard assay cannot be compared withthe others, because they have been calculated by reference to anInternational Standard, which cannot be used for the other assays.

TABLE 3 Serum IgG levels Recombinant GST-fusion proteins sample (IU/ml)Tx-15.11 Tx-1.11 Tx-1.16 Tx-4.18 Tx-2.a Tx-11.b EC2 EC3 EC4 T1 >300 7.43.5 9.8 3.7 18.3 12.5 11.0 10.0 7.6 T2 188 8.7 7.4 2.4 5.1 18.8 4.4 26.023.5 20.0 T3 265 7.4 2.8 4.6 2.8 8.4 5.6 20.4 17.9 34.0 T4 4090 23.327.8 22.4 29.7 25.1 27.4 33.6 43.3 40.5 T5 >300 12.4 9.7 25.7 2.9 nd nd30.6 34.4 19.5 T6 35 1.2 0.8 1.0 1.3 0.9 1.4 1.4 5.7 1.7 T7 58 1.0 1.30.6 2.3 1.2 1.0 4.6 5.1 1.2 T8 101 2.5 0.9 2.5 2.8 11.2 2.9 17.0 4.830.9 T9 88 16.2 5.9 2.4 2.7 6.8 6.7 9.4 27.4 29.9 T10 188 1.7 1.2 4.43.1 1.7 1.3 4.4 3.6 3.3 T11 530 28.9 12.7 12.1 31.4 32.1 31.2 32.1 42.641.5 T12 89 0.9 nd 4.8 nd 1.2 0.5 1.7 2.4 2.1 T13 1095 8.6 2.3 3.4 5.9nd nd 31.7 23.4 20.3 T14 248 12.6 4.3 2.1 0.9 nd nd 7.4 14.6 9.2 T15 15517.5 1.0 2.8 3.4 nd nd 6.7 20.1 13.0 T16 427 4.5 3.9 5.0 2.7 nd nd 17.437.9 18.9 T17 236 12.1 4.8 6.1 3.7 nd nd 22.0 33.9 12.7 T18 46 2.1 2.02.6 2.2 nd nd 1.9 5.4 1.4 T19 247 2.3 2.5 3.6 0.5 nd nd 3.7 3.9 2.3 T20100 nd nd nd nd 7.5 1.4 13.1 5.2 14.6 T21 27 2.6 0.6 4.1 1.0 nd nd 3.14.8 5.5 T22 >300 28.6 5.1 4.6 19.9 nd nd 14.5 14.8 7.4 T23 92 1.7 1.11.3 0.8 0.7 0.8 6.9 4.5 6.4 T24 68 0.5 2.1 2.0 2.6 1.3 1.3 2.5 6.6 1.4T25 63 2.4 2.4 5.5 6.8 8.8 7.7 13.8 16.6 32.9 T26 299 12.1 10.0 6.5 4.6nd nd 10.0 13.5 6.0 T27 108 3.4 4.0 5.7 3.0 nd nd 4.8 6.2 3.1 T28 68 1.72.7 3.3 2.1 2.0 2.3 6.4 6.9 7.7 T29 >300 1.0 1.1 2.8 1.2 4.0 6.3 9.113.7 23.5 T30 114 1.8 2.8 19.3 3.8 3.9 2.0 13.0 13.3 13.6 T31 35 3.2 1.23.5 0.9 1.5 1.0 3.6 6.3 5.7 T32 300 4.8 16.0 17.3 20.9 nd nd 31.6 33.18.4 T33 123 5.6 2.6 3.3 4.5 20.4 2.8 25.8 21.0 36.6 T34 60 nd nd nd nd6.2 4.2 9.1 7.7 9.3 T35 155 0.7 2.4 0.5 2.3 1.8 1.1 7.3 10.4 9.5 T36 450.3 1.6 1.1 0.8 1.2 0.9 3.2 3.2 3.1 N1 0.4 0.4 0.7 0.7 0.5 0.3 0.4 0.70.5 N2 0.7 0.5 0.5 0.4 nd nd 0.4 0.5 0.4 N3 nd nd nd nd nd nd 0.9 0.50.6 N4 nd nd nd nd nd nd 0.4 0.5 0.7 N5 nd nd nd nd 0.5 0.7 0.4 0.5 0.5N6 nd nd nd nd nd nd 0.4 0.5 0.5 N7 0.4 0.4 0.6 0.7 nd nd 1.0 0.5 0.7 N80.4 0.5 0.5 0.5 nd nd 0.4 0.5 0.6 N9 0.4 0.6 0.7 0.5 nd nd 0.4 0.5 0.4N10 nd nd nd nd nd nd 0.3 0.5 0.4 N11 nd nd nd nd nd nd 0.4 0.5 0.5 N12nd nd nd nd nd nd 0.5 0.7 0.8 N13 nd nd nd nd nd nd 0.4 0.7 0.3 N14 0.70.5 0.5 0.5 0.9 0.7 0.5 0.5 0.5 N15 0.4 0.6 0.4 1.0 0.5 0.8 0.4 0.5 0.4N16 0.8 0.2 0.5 0.6 0.5 0.4 0.5 0.7 0.5 N17 0.8 0.5 0.9 0.4 0.6 0.7 0.40.5 0.6 N18 0.3 0.4 0.7 0.5 0.6 0.6 0.4 0.6 0.9 N19 0.4 0.5 0.5 0.8 ndnd 0.5 0.8 0.8 N20 0.6 0.6 0.4 0.6 0.7 0.5 0.8 0.7 1.0 N21 0.7 0.7 0.60.5 0.5 0.4 0.4 0.7 0.6 N22 0.4 0.5 0.7 0.5 0.5 0.7 0.4 0.6 0.3 N23 0.50.5 0.7 0.5 nd nd 0.4 0.6 0.5 N24 nd nd nd nd 0.5 0.5 0.5 0.7 0.7 N25 ndnd nd nd 0.8 0.5 0.8 0.6 0.5 N26 nd nd nd nd 0.5 0.6 0.4 0.6 0.4 N27 ndnd nd nd 0.4 0.6 0.8 1.0 0.4

The following Table 4 summarizes the results of the ELISA assays basedon recombinant proteins, employing serum samples from T. gondiiseropositive and seronegative humans. In each column are reported thenumber and the corresponding percentages of reactive sera. From Table 4it clearly results that the sensitivity of the assay (see the 2^(nd)column reporting the occurrence of false negatives) is improved whenusing the chimeric antigens of the invention. This improvement isevident with respect to both the use of single antigenic fragments andthe use of a collection or mixtures (Mix-Tx-1.16/Tx-4.18/Tx-2.a,Mix-Tx-1.16/Tx-4.18/Tx-2.a, Mix-Tx-1.16/Tx-2.a/Tx-11.b) of the singledifferent antigenic fragments.

TABLE 4 Sera from T. gondii Sera from T. gondii GST-fusian proteininfected subjects uninfected subjects Tx-15.11 28/34 (82.4%) 0/15Tx-1.11 29/33 (87.9%) 0/15 Tx-1.16 31/34 (91.2%) 0/15 Tx-4.18 27/33(81.8%) 0/15 Tx-2.a 21/23 (91.3%) 0/14 Tx-11.b 18/23 (78.3%) 0/14Mix-Tx-1.16/Tx-4.18/Tx-2.a 35/36 (97.2) 0/27 Chimera EC2 36/36 (100%)0/27 Mix-Tx-15.11/Tx-1.11/ 35/36 (97.2%) 0/27 Tx-11.b Chimera EC3 36/36(100%) 0/27 Mix-Tx-1.16/Tx-2.a/Tx-11.b 34/36 (94.4%) 0/27 Chimera EC436/36 (100%) 0/27Immunoreactivity of Single Antigenic Domains within the ChimericRecombinant Antigens with IgG Antibodies of Sera from T. gondii InfectedIndividuals

To verify that the chimeric antigens retain the immunoreactivity of thesingle antigen fragments used for their construction, human sera thatspecifically reacted, in ELISA assays, with single antigen fragments,were adsorbed with different combinations of single antigens and thenassayed with the chimeric proteins. To this aim, distinct combinationsof the antigen fragments, expressed as GST-fusion products, were coatedonto Maxisorb-multiwells plates (Nunc) at a concentration of 10 μg/ml incoating buffer (50 mM NaHCO₃, pH 9.6) and then incubated overnight at 4°C. The plates were extensively washed and subsequently incubated for 30min. at 37° C. with serum samples (20 μl/well) in blocking solution (5%non-fat dry milk, 0.05% Tween-20 in PBS). The fragment-specificantibody-depleted sera were recovered from each well, added to a newwell, incubated for 30 min., and the same procedure was repeated 6 moretimes. Samples that have been depleted for specific antibodies against asingle or multiple antigen fragments were finally analyzed by ELISAassays on the chimeric antigens. For this purpose, the chimeric antigensEC2, EC3 and EC4, as GST-fusion products, were coated overnight at 4° C.onto Maxisorb-multiwells plates at a concentration of 5 μg/ml. Thecoated plates were blocked and subsequently incubated for 1 h at 37° C.with the antibody-depleted human sera diluted 1:100 in blockingsolution. Plates were extensively washed and anti-human-IgG alkalinephosphatase-conjugated antibodies (Sigma-Aldrich, USA), diluted 1:7500in blocking solution, was then added to each well. Finally, thechromogenic substrate p-nitrophenyl phosphate (Sigma-Aldrich, USA)revealed the enzymatic activity. The results were recorded as thedifference between the absorbance at 405 and 620 nm, detected by anautomated ELISA reader (Labsystem Multiskan, Finland). For each samplethe assay was done in duplicate and average values were calculated.

Biochemical Modification of EC2 and EC3 Chimeric Antigens

To analyze the immunoreactivity of the chimeric antigens EC2 and EC3with specific anti-Toxoplasma IgM antibodies in patient sera, therecombinant proteins were chemically modified by biotinylation. To thisaim, the purified GST-EC2 and GST-EC3, diluted at a concentration of 1mg/ml in PBS were incubated in the presence of a five-fold molar excessof sulfosuccinimidyl-6-(biotin-amido)hexanoate (Sulfo-NHS-LC-Biotin fromPierce, USA) for 3 hours on ice. The proteins were then dialyzedovernight against PBS to remove excess of non-reacted and hydrolyzedbiotin reagents. Levels of biotin incorporation into chimeric antigenswas determined by using “EZ Biotin Quantitation Kit” (Pierce, USA),resulting in 1.4 biotin/molecule for GST-EC2 and 1.3 biotin/molecule forGST-EC3. The biotin-labeled products were finally diluted at aconcentration of 0.5 mg/ml and stored at −20° C. until use.

Immunoreactivity of the Biotin-Labeled EC2 and EC3 withToxoplasma-Specific IgM Antibodies: IgM Rec-ELISA

To investigate the immunoreactivity of recombinant antigens withToxoplasma-specific immunoglobulins M, a double-sandwich immunoassay wasemployed (IgM Rec-ELISA). Maxisorb plates (Nunc, USA) were coated withanti-human IgM antibodies (Sigma-Aldrich, USA) at a concentration of 10μg/ml in coating buffer (50 mM NaHCO₃, pH 9.6). Plates were blocked with3% bovine serum albumin in PBS (blocking solution) for 1 h at 37° C. andsubsequently incubated for 1 h at 37° C. with serum samples in blockingsolution. Plates were washed and then incubated for 2 h at roomtemperature with the biotin-labeled GST-fusion proteins, diluted inblocking solution. After being extensively washed the plates wereincubated for 1 h at room temperature with horseradishperoxidase-conjugated streptavidin (Pierce, USA) at a concentration of 1μg/ml in blocking solution. Finally, the enzymatic activity was revealedincubating plates for 30 min. at room temperature with the substratetetramethylbenzidine (Sigma-Aldrich, USA). Results were recorded as thedifference between the absorbance at 450 and 620 nm, detected by anautomated ELISA reader (Labsystem Multiskan, Finland). For each samplethe assay was done in duplicate and average values were calculated.

Thermal Stability of the Biotin-Labeled Chimeric Antigens

In order to determine the thermal stability of the biotin-labeledGST-EC2 and GST-EC3, recombinant products were diluted at aconcentration of 5 μg/ml in the commercial buffer “Stabilzyme”(SurModics, USA) and stored at +4° C. until use. After differentinterval times (up to 80 days), the immunoreactivity of recombinantproteins in the IgM Rec-ELISA analysis was assessed and results obtainedanalyzing the corresponding products maintained frozen at −20° C. werecompared. The IgM Rec-ELISA was performed as described above, using thebiotin-labeled GST-EC2 and GST-EC3 antigens at a final concentration of500 ng/ml in blocking solution (3% BSA in PBS) and human sera diluted1:100 in blocking solution. For each sample the assay was done induplicate and average values were calculated. The ID50, calculated asday-limit when the 50% of toxoplasma-specific IgM-immunoreactivity wasmeasured, were 189 days and 97 days for GST-EC2 and GST-EC3,respectively. These findings clearly indicate that the chimeric antigensof the invention are stable in diluted solutions for a long time, whicha fundamental requisite for the commercial usefulness of a recombinantproduct.

Expanded Evaluation of IgM Rec-ELISA

The biotin-labeled GST-EC2 and GST-EC3 chimeric antigens were assayedwith IgM antibodies in sera from T. gondii infected individuals and theresults of the IgM Rec-ELISAs were compared with those obtained withcommercial assays employing lysed, whole-cell Toxoplasma antigen (VIDASsystem from bioMerieux, France; ETI-TOXOK-M Reverse-PLUS from DiaSorin,Italy). To this aim, serum samples from women who acquired primarytoxoplasmosis during gestation and referred for post-natal follow-up atthe Center for Perinatal Infection of Campania Region, Italy, wereassayed. The bioMerieux VIDAS Toxo IgG and IgM assays were used toselect three groups of serum samples for the Toxoplasma IgM Rec-ELISAperformance evaluation. Group A (n=22) was composed of samples negativefor T. gondii-specific IgM and IgG antibody as measured by the VIDASToxo IgM and IgG assays. Group B (n=18) was composed of samples with aserological profile consistent with a chronic infection (presence of T.gondii-specific IgG antibody and absence of T. gondii-specific IgM asmeasured by the VIDAS Toxo IgM and IgG assays, respectively). Group C(n=50) was composed of samples with a serological profile consistentwith an acute infection (presence of T. gondii-specific IgM and IgGantibodies as measured by the VIDAS Toxo IgM and IgG assays). IgM RecELISA was performed as described above and for each serum sample theassay was done in duplicate and average values were calculated.

The following Table 5 shows the IgM reactivity of the biotin-labeledGST-EC2 and GST-EC3 chimeric antigens, compared to the results obtainedwith commercial assays (VIDAS and ETI-TOXO-K), by using sera from groupA (A1-A22), group B (B1-B18) and group C (C1-C50). TheToxoplasma-specific IgG levels, calculated as International Units (IU)are also reported. For each biotin-labeled GST-fusion product thecut-off was determined as the mean plus two times the standard deviationof the absorbency readings obtained from the T. gondii-specific IgMnegative sera (groups A and B, n=40). Cut-off values for VIDAS IgM,ETI-TOXOK-M Reverse-PLUS, GST-EC2 and GST-EC3 were 0.650, 0.500, 0.343and 0.378, respectively. Values typed in bold indicate a positiveresponse.

TABLE 5 Toxo-IgG VIDAS ETI-TOXOK-M IgM Rec-ELISA Serum (UI/ml) IgMReverse-PLUS GST-EC2 GST-EC3 A1 0 0.05 0.397 0.268 0.256 A2 4 0.22 0.3170.263 0.270 A3 0 0.18 0.252 0.264 0.237 A4 0 0.05 0.375 0.324 0.241 A5 20.17 0.272 0.298 0.222 A6 0 0.03 0.288 0.270 0.234 A7 0 0.19 0.210 0.2150.379 A8 0 0.10 0.108 0.203 0.296 A9 0 0.06 0.324 0.314 0.291 A10 0 0.090.339 0.325 0.286 A11 0 0.05 0.193 0.223 0.271 A12 2 0.08 0.134 0.2680.378 A13 4 0.12 0.115 0.309 0.335 A14 0 0.23 0.115 0.221 0.286 A15 00.06 0.230 0.281 0.374 A16 2 0.08 0.132 0.317 0.269 A17 1 0.18 0.1230.277 0.281 A18 0 0.35 0.097 0.316 0.279 A19 0 0.28 0.346 0.274 0.272A20 0 0.09 0.054 0.259 0.132 A21 0 0.61 0.206 0.24 0.312 A22 0 0.060.127 0.238 0.233 B1 24 0.06 0.239 0.255 0.189 B2 10 0.09 0.076 0.2830.304 B3 44 0.14 0.124 0.265 0.261 B4 44 0.28 0.195 0.298 0.216 B5 250.1 0.131 0.273 0.185 B6 57 0.12 0.164 0.296 0.293 B7 12 0.22 0.1850.257 0.194 B8 58 0.12 0.148 0.255 0.248 B9 56 0.4 0.174 0.232 0.268 B1019 0.09 0.068 0.290 0.194 B11 56 0.16 0.136 0.179 0.221 B12 45 0.120.139 0.235 0.181 B13 87 0.12 0.096 0.207 0.218 B14 27 0.15 0.144 0.1960.174 B15 33 0.46 0.242 0.285 0.378 B16 13 0.04 0.064 0.161 0.177 B17 670.13 0.111 0.177 0.213 B18 53 0.27 0.170 0.238 0.165 C1 28 5.37 1.040.350 0.548 C2 255 3.86 1.49 0.546 0.498 C3 78 3.01 1.38 0.471 0.867 C41358 2.28 1.17 0.464 0.453 C5 178 2.31 1.27 0.598 0.406 C6 155 2.00 0.970.993 0.720 C7 109 3.20 1.76 0.794 0.642 C8 99 3.16 1.78 0.572 0.389 C9103 2.28 1.34 1.056 1.222 C10 85 2.22 1.44 0.930 0.704 C11 70 1.01 0.790.416 0.376 C12 26 1.34 0.93 0.392 0.461 C13 36 1.22 0.86 0.532 0.499C14 156 0.99 0.58 0.534 0.833 C15 204 0.93 0.90 0.810 0.710 C16 133 1.130.85 0.465 0.322 C17 183 1.14 0.82 0.320 0.327 C18 242 0.90 0.71 0.4970.500 C19 80 1.00 0.79 0.444 0.706 C20 258 1.40 0.88 2.678 0.484 C21 2781.69 1.06 0.703 0.509 C22 246 1.25 0.76 1.094 0.780 C23 59 1.23 0.710.495 1.499 C24 38 0.78 0.87 0.584 0.455 C25 130 0.76 0.92 0.562 0.545C26 262 0.84 0.65 0.649 0.551 C27 168 0.96 0.85 1.439 0.938 C28 126 0.780.80 2.475 1.160 C29 197 1.38 0.61 0.544 0.358 C30 127 0.86 0.52 0.8470.531 C31 72 1.28 0.93 1.756 0.891 C32 130 0.77 0.71 0.505 0.381 C33 4391.00 0.66 0.834 0.464 C34 83 0.66 1.32 1.162 0.989 C35 178 0.89 0.870.694 0.487 C36 560 0.86 0.69 0.817 0.628 C37 223 0.96 0.73 0.531 0.819C38 242 0.98 0.41 0.379 0.318 C39 118 1.16 0.84 0.420 0.380 C40 232 1.391.01 0.490 0.467 C41 213 1.03 1.05 0.750 0.822 C42 243 1.06 0.97 0.5340.502 C43 154 0.75 0.73 0.455 0.337 C44 35 1.90 1.51 0.383 1.008 C45 6670.85 1.01 0.366 0.285 C46 275 0.95 0.99 0.411 0.544 C47 157 1.93 1.360.382 0.464 C48 1037 1.08 0.51 0.385 0.301 C49 92 1.31 0.68 0.537 0.427C50 255 0.69 0.69 0.354 0.801

The following Table 6 shows the performance characteristics of thecommercial assays (VIDAS IgM and ETI-TOXOK-M Reverse PLUS), compared tothe results obtained with the biotin-labeled EC2 and EC3 chimericantigens (IgM Rec-ELISA). From Table 6 it clearly results that bothspecificity and positive predictive values of the assays (see the 3^(rd)column reporting the occurrence of false positives) reached the maximum(100%) when using the chimeric antigens of the invention. With regard tosensitivity and agreement, both the commercial test ETI-TOXOK-Memploying lysed, whole-cell Toxoplasma antigen and the IgM rec-ELISAwith the chimeric antigen EC2 display identical performancecharacteristics, with both values very close to 100%.

TABLE 6 Diagnostic Sensitivity Specificity Agreement PPV* NPV* test (%)(%) (%) (%) (%) VIDAS IgM 100 100 100 100 100 ETI-TOXOK-M 98.0 100 98.9100 97.6 EC2-IgM 98.0 100 98.9 100 97.6 Rec-ELISA EC3-IgM 84.0 100 91.1100 83.3 Rec-ELISA *PPV, positive predictive value; NPV, negativepredictive value.

Finally, the immunoreactivity of the biotin-labeled GST-EC2 and GST-EC3antigens with IgM antibodies in sera from infants with congenitaltoxoplasmosis was investigated. In a retrospective study, sera from 30infants of mothers with primary T. gondii infection during pregnancywere analyzed. Twenty infants had congenital toxoplasmosis and ten wasuninfected, as demonstrated by the persistence or disappearance ofToxoplasma-specific IgG antibodies after 12 months of age, respectively.Within the infected patient cohort, the gestational age at the time ofmaternal infection was the second trimester in 6 mothers and the thirdtrimester in 14 mothers. 30 serum samples from infected and uninfectedinfants were analyzed by IgM Rec-ELISA, and results obtained withcommercial assays employing the whole-cell Toxoplasma antigen (ELFA-IgMand ETI-TOXOK-M Reverse PLUS) were compared. Specific levels ofanti-Toxoplasma IgG ranged from 28 to 1147 IU/ml for sera from infectedinfants and from 19 to 170 IU/ml for sera from uninfected subjects. Forevery GST-fusion product the cut-off value was determined as the meanplus 2SD of the absorbency readings obtained with sera from uninfectedinfants. In Table 7 are summarized the results of the IgM Rec-ELISAswith individual sera from infected infants. Overall, the number ofIgM-reactive sera ranged from 70% (14/20) to 50% (10/20) using theGST-EC2 and GST-EC3 antigens, respectively. In contrast, only 7 out of20 infected infants (35%) had positive results when ELFA-IgM orETI-TOXOK-M assays were employed. Among uninfected infants, none of thesera recognized GST-EC2 and GST-EC3 antigens in the IgM Rec-ELISA orresulted to be positive using commercial assays.

In conclusion, these results demonstrate that the use of recombinantchimeric antigens is effective in distinguishing T. gondii-infected fromuninfected individuals, having comparable or even better assayperformance with respect of using the whole-cell tachyzoite antigen, andcould provide the basis for standardized commercial immunoassays fortoxoplasmosis serodiagnosis.

TABLE 7 Toxoplasma-specific IgM reactivity of serum samples from 30infants born to mothers with primary T. gondii infection acquired duringpregnancy^(a) Time IgM Rec-ELISA after IgG ELFA- ETI- cutoff^(d) Patientbirth levels IgM ToxoM GST- GST- no. (wk) Onset^(b) (IU/ml) cutoff^(c)cutoff^(c) EC2 EC3 T1 1 B 169 6.41 2.66 2.479 0.542 T2 2 B 988 0.73 0.150.360 0.270 T3 2 Sub 300 0.09 0.13 0.212 0.209 T4 3 Sub 57 0.05 0.230.206 0.211 T5 3 Sub 124 0.13 1.62 0.641 1.103 T6 4 Sub 218 0.04 0.090.452 0.269 T7 4 S 157 2.61 1.62 1.522 0.225 T8 4 S 172 3.98 1.50 1.8040.353 T9 5 S 1147 0.07 0.10 0.519 0.206 T10 5 B 47 0.11 0.12 0.272 0.276T11 6 Sub 28 0.10 0.18 2.617 0.731 T12 6 Sub 136 0.07 0.11 0.314 0.216T13 7 S 209 0.88 0.47 0.683 0.217 T14 8 Sub 43 0.06 0.07 0.196 0.213 T158 B 160 0.82 0.08 0.206 0.219 T16 8 B 64 0.02 0.40 0.231 0.228 T17 8 Sub145 0.31 0.57 0.985 0.314 T18 9 Sub 300 6.37 1.30 0.548 0.315 T19 12 Sub196 0.05 0.17 0.463 0.268 T20 12 Sub 75 0.05 0.07 0.237 0.222 N1 5 900.38 0.06 0.237 0.218 N2 5 170 0.06 0.07 0.204 0.200 N3 5 66 0.23 0.090.238 0.235 N4 3 44 0.07 0.12 0.176 0.209 N5 9 41 0.05 0.05 0.209 0.193N6 5 66 0.07 0.06 0.194 0.196 N7 8 13 0.09 0.06 0.193 0.201 N8 9 13 0.140.06 0.208 0.231 N9 6 19 0.28 0.07 0.184 0.215 N10 5 20 0.13 0.05 0.1890.240 Notes to Table 7 ^(a)Serum samples from T. gondii infected(T1-T20) or uninfected children (N1-N10) were analyzed by IgM Rec-ELISAswith GST-EC2 and GST-EC3 antigens or by commercial assays (ELFA-IgM andETI-TOXO-M). ^(b)Severity of clinical onset: S. severe; B. benign; Sub.subclinical. ^(c)Cutoff values for the ELFA-IgM and ETI-TOXO-M assayswere 0.65 and 0.41 as indicated by manufacturers. respectively. Boldtype. values > cutoff. ^(d)Cutoff values for the IgM Rec-ELISA usingGST-EC2 and GST-EC3 antigens were 0.25 and 0.26. respectively. Boldtype. values > cutoff.

1. A chimeric recombinant antigen containing a fusion of at least threedifferent antigenic regions of Toxoplasma gondii, wherein said antigenicregions are B-cell epitopes, which bind to Toxoplasma gondii-specificantibodies.
 2. The chimeric antigen of claim 1, wherein the Toxoplasmagondii-specific antibodies are extracted from sera of subjects who havebeen infected by Toxoplasma gondii.
 3. The chimeric antigen of claim 1,wherein the three different antigenic regions are linked by a covalentbond or by a peptide linker.
 4. The chimeric antigen of claim 1, whereinthe three different antigenic regions have an amino acid sequenceselected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:
 42. 5. Thechimeric antigen of claim 1, comprising the amino acid sequence of SEQID NO: 28,
 6. The chimeric antigen according to claim 1, comprising theamino acid sequence of SEQ ID NO:
 30. 7. The chimeric antigen accordingto claim 1, comprising the amino acid sequence of SEQ ID NO:
 32. 8. Anucleotide sequence that codes for the chimeric antigen according toclaim 1 or that hybridizes under stringent hybridization conditions witha nucleotide sequence coding for the chimeric antigen according toclaim
 1. 9. The nucleotide sequence according to claim 8, comprising atleast three different nucleotide sequences selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9 and SEQ ID NO:
 11. 10. The nucleotide sequence according toclaim 8, comprising the nucleotide sequence of SEQ ID NO:
 27. 11. Thenucleotide sequence according to claim 8, comprising the nucleotidesequence of SEQ ID NO:
 29. 12. The nucleotide sequence according toclaim 8, comprising the nucleotide sequence of SEQ ID NO:
 31. 13.(canceled)
 14. A chimeric recombinant antigen encoded by a nucleotidesequence that hybridizes under stringent hybridization conditions with anucleotide sequence coding for the chimeric antigen according toclaim
 1. 15. The nucleotide sequence of claim 8, which is a DNAsequence.
 16. The nucleotide sequence of claim 15, wherein the DNAsequence is comprised in a vector.
 17. The nucleotide sequence of claim16, wherein the vector is comprised in a host cell transformed with saidvector.
 18. The chimeric recombinant antigen according to claim 1,obtained by a process comprising culturing the host cell of claim 17 andisolating the chimeric antigen from the cultured host cells.
 19. Amethod for the diagnosis of Toxoplasma gondii infections, the methodcomprising contacting a chimeric antigen according to claim 1 with abody component of a subject for a time and under condition to allow thechimeric antigen to bind an antibody present in the body component in anantigen/antibody immune complex and detecting the antigen/antibodyimmune complex comprising the chimeric antigen.
 20. The method accordingto claim 19 for the diagnosis of congenital toxoplasmosis in infants.21. The method according to claim 19 for the diagnosis of the time ofinfection.
 22. A kit for the diagnosis of Toxoplasma gondii infection,containing at least one chimeric antigen according to claim
 1. 23. Thekit of claim 22, wherein the kit is for the diagnosis of an acute and/orchronic Toxoplasma gondii infection. 24-32. (canceled)